Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils

International Journal of Molecular Sciences

Article Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils

Mantas Ziaunys, Andrius Sakalauskas , Kamile Mikalauskaite, Ruta Snieckute and Vytautas Smirnovas *

Life Sciences Center, Institute of Biotechnology, Vilnius University, LT-10257 Vilnius, Lithuania; [email protected] (M.Z.); [email protected] (A.S.); [email protected] (K.M.); [email protected] (R.S.) * Correspondence: [email protected]

Abstract: Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the

resulting ﬁbril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one ﬁbril type and that this variability

Citation: Ziaunys, M.; Sakalauskas, may depend on the state of the initial prion protein molecules. A.; Mikalauskaite, K.; Snieckute, R.; Smirnovas, V. Temperature- Keywords: amyloids; prion proteins; protein aggregation; fibril structure Dependent Structural Variability of Prion Protein Amyloid Fibrils. Int. J. Mol. Sci. 2021, 22, 5075. https:// doi.org/10.3390/ijms22105075 1. Introduction Amyloidogenic protein aggregation into insoluble, beta-sheet rich fibrils is linked with Academic Editor: the onset of several neurodegenerative disorders, such as Alzheimer’s, Parkinson’s or prion Vladimir N. Uversky diseases [1,2]. The way these structures form and propagate is still not fully understood, as evidence for new aggregation mechanisms or fibril structural features [3–5] keeps appearing Received: 20 April 2021 on a regular basis. This lack of crucial information is likely one of the factors that has led Accepted: 9 May 2021 Published: 11 May 2021 to countless failed clinical trials [6] and to only a handful of effective, disease-modifying drugs [7]. Considering that amyloid diseases affect millions of people worldwide and the

Publisher’s Note: MDPI stays neutral number is expected to continuously increase [8,9], it is of vital importance to gain a better with regard to jurisdictional claims in understanding of the intricacies of amyloid aggregation. published maps and institutional affil- One of the more interesting aspects of amyloid aggregation is the ability of a single iations. type of protein to associate into multiple conformationally-distinct fibrils [10]. Such a phenomenon was observed with prion proteins, both in vivo and in vitro [11–14] and later– with other amyloidogenic proteins, such as amyloid beta [15], alpha-synuclein [16,17] and insulin [18,19]. These distinct conformation fibrils possess specific replication rates [20,21], morphologies [22], secondary structures [16,23] and stabilities [24]. In addition, some of Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. these parameters are either codependent or change even after fibrils have been formed. This article is an open access article It has been shown using computational methods that there is a correlation between the distributed under the terms and rate of self-replication and fibril symmetry, and stability [25,26]. It was also observed conditions of the Creative Commons that oligomeric or protofibrillar intermediate aggregates gain mechanical stability when Attribution (CC BY) license (https:// converting to fully formed fibrils [27]. creativecommons.org/licenses/by/ Such a variation in their properties is likely one of the main aspects why potential 4.0/). drug candidates appear effective under a certain set of conditions, while being completely

Int. J. Mol. Sci. 2021, 22, 5075. https://doi.org/10.3390/ijms22105075 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5075 2 of 16

inefficient in others [28]. It has been observed in multiple studies that the conformation of resulting fibrils depends highly on the environmental conditions, under which the protein is aggregated [29,30]. These conditions include temperature [31], agitation [32], pH [18], ionic strength [33], denaturant concentration [14] and protein concentration [34]. In our recent study, we have also shown that prion proteins are capable of forming two distinct conformation fibrils under identical conditions [23]. This hints at a possibility that primary nucleation, a process during which the initial aggregation centers from, may randomly generate distinct nuclei, only some of which can further propagate under the given environmental conditions. Determining conformational differences between amyloid fibrils is typically done by evaluating their morphological features [35], such as aggregate height, width, length and periodicity patterns, and by acquiring information about their secondary structure [36]. The methods used for this, namely atomic force microscopy (AFM) [35] and Fourier-transform infrared spectroscopy (FTIR) [37], are difficult to apply in situations, where a large number of samples have to be differentiated. In recent years, both high-throughput screening platforms [38] and computer-aided molecule design systems [39] have advanced enough to identify neurodegenerative-disease related protein–ligand interactions and possible mechanisms of fibril formation. However, since protein aggregation is still not fully understood, such methods are not ideal for determining the highly complex structure of fully formed aggregates. As an alternative, the selective and conformation-specific binding of a fluorescent probe, thioflavin-T (ThT) [23,40,41], may be used to detect different types of aggregates. Such a method was applied in our aforementioned study with prion proteins and it was efficient at identifying different types of insulin fibrils as well [40]. Applying this type of initial screening would allow to sort out fibril samples with distinct conformations in a much larger scale assay. Prion protein aggregation experiments are conducted under a variety of conditions in vitro, ranging from ambient temperature [42] to well-above physiological temperatures [43]. In order to determine if this environmental factor has an effect on prion protein fibril variability, we tracked the aggregation of a mouse prion protein under a range of different temperatures, using a large identical sample size to evaluate their seemingly random, conformational variations. To achieve complete fibrillization in a reasonable time frame, vigorous, fragmentation-inducing agitation was also used for every temperature condition. A ThT-assay was then employed as an initial means of sorting the distinct fibril types, which were then examined using FTIR and AFM.

2. Results A large sample size of mouse prion protein fragment (89–230) was aggregated under a range of temperatures (from 25 to 65 ◦C) with all other conditions, such as buffer solution, protein concentration and volume remaining identical. Plotting the lag time (tlag) dependence on aggregation temperature (Figure1A) revealed a discontinuity in the linear trend at 45 ◦C. This is caused by the protein transitioning from being in mostly folded states (at temperatures below 45 ◦C) and mostly unfolded (above 45 ◦C) [43]. This is further supported by visualizing the data in an Arrhenius plot (Figure1B), where the activation of nuclei formation was (78.7 ± 7.0) kJ/mol at low and (30.0 ± 5.0) kJ/mol at high temperatures. This transition temperature is in line with previously reported data, [43] where a shift in activation energy was also observed. Int. J. Mol. Sci. 2021, 22, x 3 of 18

way analysis of variance (ANOVA) statistical analysis of the data (n = 90 for each case) using a Bonferroni means comparison (significance level–0.01) revealed that there was a difference between the 35–45 °C sample and 50–60 °C sample fluorescence intensity distributions, where 35–45 °C fibril-bound ThT possesses a considerably larger average signal intensity. Interestingly, there was no significant difference between the lowest and highest temperature sample sets (significance level–0.01), apart from the 25 °C samples having a greater standard deviation due to the existence of a few high fluorescence intensity samples. Both of these aspects could be explained by a larger fibril concentration or the formation of superstructural fibril assemblies, however, that is not the case. Each sample contained an identical concentration of prion protein and their aggregation kinetic curves reached a plateau, indicating a finished fibrillization process. Afterwards, each sample was diluted with the reaction buffer (containing 100 µM ThT) and sonicated, which removes the possibility of large aggregate structures entrapping the dye molecules Int. J. Mol. Sci. 2021, 22, 5075 [44] or certain samples having hydroxylated ThT [45]. This leaves the option of3 different of 16 samples containing distinct fibrils, which have conformation-specific dye-binding.

-3.5 A B 600 C 1000 -4.0 500 -4.5 500 -5.0 400

) (30.0 ± 5.0) kJ/mol -1

lag 300 (min) -5.5 200 lag ln(t t -6.0 200

100 -6.5 100 (78.7 ± 7.0) kJ/mol -7.0

50 (a.u.)Fluorescence intensity 0 -7.5 25 30 35 40 45 50 55 60 65 3.4 3.3 3.2 3.1 3.0 2.9 25 30 35 40 45 50 55 60 65 3 -1 Temperature (°C) 10 /T (K ) Temperature (°C)

FigureFigure 1. Prion 1. Prionprotein protein aggregation aggregation tlag and tlag andfibril-bound fibril-bound thioflavin-T thioflavin-T (ThT) (ThT) fluore fluorescencescence intensity intensity dependence on on the the ag- gregationaggregation reaction reaction temperature. temperature. tlag dependence tlag dependence on the on reaction the reaction temperature temperature (A) and (A) andthe same the same data data displayed displayed in inan an Arrhe- nius plotArrhenius (B), with plot (reactionB), with reactionactivation activation energies energies shown shown below below lin linearear fit fit curves. curves. Sonicated and and diluted diluted fibril-ThT fibril-ThT sample sample fluorescencefluorescence intensity intensity distribution distribution (at 25 (at °C) 25 ◦ C)dependence dependence on on the the reaction reaction temperature temperature (C).). EachEach samplesample set set contained contained 90 data points,90 data obtained points, obtained by tracking by tracking the aggregation the aggregation and and fluorescence fluorescence intensity intensity ofof 9090 prion prion protein protein aggregation aggregation reactions reactions simultaneouslysimultaneously at a set at a temperature. set temperature. In InA Aand and C Cgraphs, graphs, box box plots plots indicate the the interquartile interquartile range range and and error error bars bars are for are for −1 −1 one standardone standard deviation. deviation. Graph Graph B points B points are are the the average average ln(t ln(tlaglag) values) values calculated calculated from from the the t lag valuesvalues and errorerror bars bars are one standardare one standarddeviation. deviation. Dashed Dashedblue lines blue correspond lines correspond to the to temperature the temperature at which at which a non-linear a non-linear change change in tlaglag and ThT fluorescenceThT fluorescence intensity intensityis observed. is observed. Red lines Red (B)lines are linear (B) are fits linear of 25 fits – of45°C 25–45 and◦C 45 and – 65°C 45–65 temperature◦C temperature data data points. points. Aggre- gationAggregation tlag box plots tlag (A)box and plots their (A) respective and their respective sample fluorescence sample fluorescence intensities intensities (C) are (C )colour-coded. are colour-coded.

In orderWhen examiningto separate the and fluorescence identify distinct intensity fibril of fibril-bound samples, ThTan excitation–emission molecules (when all ma- samples were cooled down to 25 ◦C; Figure1C), two interesting aspects are observed. First, trix (EEM) of each sample‘s fibril-bound ThT was scanned. The fluorescence emission in- there was a significantly larger amount of high fluorescence intensity samples (300–600 a.u.) tensitywhen “centers the protein of mass“ was aggregated were then at temperaturescompared for below all temperature 45 ◦C, when compared conditions to higher(Figure 2). Wetemperatures, can see that wherein all onlycases, a singlethere highwas intensity a cluster sample of these was positions seen. Secondly, with therevarying was alsosize and dispersion.a discontinuity In the of case fluorescence of samples intensity from values25 to 45 occurring °C (Figure at 45 2A–E),◦C. The the one-way clusters analysis were of more compactvariance than (ANOVA) their higher statistical temperature analysis of thecounterparts data (n = 90 (Figure for each 2F–I), case) usingwith athe Bonferroni highest dis- persionmeans observed comparison in (significancethe 50 °C sample level–0.01) set (Figure revealed 2F). that there was a difference between the ◦ ◦ ◦ 35–45 C sample and 50–60 C sample fluorescence intensity distributions, where 35–45 C fibril-bound ThT possesses a considerably larger average signal intensity. Interestingly, there was no significant difference between the lowest and highest temperature sample sets (significance level–0.01), apart from the 25 ◦C samples having a greater standard deviation due to the existence of a few high fluorescence intensity samples. Both of these aspects could be explained by a larger fibril concentration or the formation of superstructural fibril assemblies, however, that is not the case. Each sample contained an identical concentration of prion protein and their aggregation kinetic curves reached a plateau, indicating a finished fibrillization process. Afterwards, each sample was diluted with the reaction buffer (containing 100 µM ThT) and sonicated, which removes the possibility of large aggregate structures entrapping the dye molecules [44] or certain samples having hydroxylated ThT [45]. This leaves the option of different samples containing distinct fibrils, which have conformation-specific dye-binding. In order to separate and identify distinct fibril samples, an excitation–emission matrix (EEM) of each sample‘s fibril-bound ThT was scanned. The fluorescence emission intensity “centers of mass” were then compared for all temperature conditions (Figure2). We can see that in all cases, there was a cluster of these positions with varying size and dispersion. In the case of samples from 25 to 45 ◦C (Figure2A–E), the clusters were more compact than their higher temperature counterparts (Figure2F–I), with the highest dispersion observed in the 50 ◦C sample set (Figure2F). Int. J. Mol. Sci. 2021, 22, 5075 4 of 16 Int. J. Mol. Sci. 2021, 22, x 4 of 18

459 459 459 A All samples 25°C B 30°C C 35°C Selected: 456 Cluster samples 456 456 Outlier samples 453 453 453 I 450 450 450 IV IV III II 447 IV 447 I 447 IV II II III II III IV I I IV I II 444 444 II 444 III III I III 441 441 441 Excitation wavelength (nm) wavelength Excitation 438 438 438 481 482 483 484 485 486 487 488 481 482 483 484 485 486 487 488 481 482 483 484 485 486 487 488

459 459 459 D IV 40°C E 45°C F 50°C

456 456 456 I I 453 453 453 III II 450 450 450

II III IV II IV II 447 IV 447 447 I I I III III II 444 444 444 II I IV III IV 441 441 441 III Excitation wavelength (nm) wavelength Excitation 438 438 438 481 482 483 484 485 486 487 488 481 482 483 484 485 486 487 488 481 482 483 484 485 486 487 488 459 G 55°C 459 H 60°C 459 I 65°C 456 456 456

453 453 453

450 III 450 III 450 IV II IV III IV III 447 IV 447 447 II III II I 444 II 444 III 444 II I II I IV I IV I 441 441 441 I Excitation wavelength (nm) wavelength Excitation 438 438 438 481 482 483 484 485 486 487 488 481 482 483 484 485 486 487 488 481 482 483 484 485 486 487 488 Emission wavelength (nm) Emission wavelength (nm) Emission wavelength (nm)

Figure 2. Prion proteinprotein fibril-boundfibril-bound thioflavin-T thioflavin-T (ThT) (ThT) fluorescence fluorescence excitation-emission excitation-emission matrix matrix “center “center of mass“of mass“ distribution. distribu- Excitation-emissiontion. Excitation-emission matrix matrix (EEM) (EEM) “centers “centers of mass“ of mass“ were calculatedwere calculated for samples for samples prepared prepared under under 25 ◦C( 25A), °C 30 (A◦C(), 30B), °C 35 (B◦C), (35C), °C 40 (◦CC(), 40D), °C 45 (◦DC(), E45), °C 50 ◦(C(E), F50), 55°C◦ (C(F),G 55), 60°C ◦(C(G),H 60) and °C ( 65H)◦ C(andI) 65 temperature °C (I) temperature conditions conditions as described as described in the Materials in the andMaterials Methods and section. Methods All section. samples All are samples marked are in marked blue, while in blue samples, while selected samples for selected further analysisfor further from analysis the main from cluster the main and cluster and outliers are marked green and red with Roman numerals respectively. Each EEM set was obtained by scanning outliers are marked green and red with Roman numerals respectively. Each EEM set was obtained by scanning 90 individual 90 individual prion protein fibril samples after they were diluted, sonicated and set to 25 °C. prion protein fibril samples after they were diluted, sonicated and set to 25 ◦C.

An important thing to notice is that each sample set contained several points, which did not belong to thethe mainmain cluster.cluster. TheseThese outliersoutliers hadhad quitequite extremeextremeexcitation/emission excitation/emission wavelength shiftsshifts whenwhen comparedcompared to to their their respective respective cluster, cluster, and and very very high high or or low low fluores- fluo- rescencecence intensity intensity values. values. In In some some of theof the sets, sets there, there were were samples samples with with 10 10 nm nm differences differences in intheir their excitation excitation wavelength wavelength (Figure (Figure2D,G). 2D,G). Since Since there there were were multiple multiple outliers outliers in each in case,each case,and differentand different fluorescence fluorescence intensity intensity samples samples within within the mainthe main cluster, cluster, four four most most distinct dis- tinctEEM EEM outliers outliers (Figure (Figure2 (red), 2 (red), Appendix AppendixA Table A A1Table) and A1) four and main four cluster main cluster samples samples from fromlowest lowest to highest to highest fluorescence fluorescence (Figure (Figure2 (green), 2 (green), Appendix AppendixA Table AA1 Table) were A1) chosen were for chosen fur- forther further structural structural examination. examinatio Eachn. sample Each sample was then was replicated then replicated to both increaseto both increase the mass the of massavailable of available aggregates, aggregates, and to test and their to self-replicationtest their self-replication propensity. propensity. In general, In no general, significant no significantvariations invariations the rate ofin replicationthe rate of werereplication observed were (Appendix observedA (Appendix, Figure A1 A,A–I), Figure however, A1A- I),most however, samples most retained samples their retained conformation-specific their conformation-specific fluorescence fluorescence emission intensities emission after in- tensitiesaggregation, after especiallyaggregation, visible especially in the casevisible of lowerin the temperaturecase of lower samples temperature (Appendix samplesA, (AppendixFigure A1). A, Figure A1). The replicated outlier (Figure 22 (red),(red), AppendixAppendixA A,, Table Table A1 A1)) and and cluster cluster ( (FigureFigure2 2 (green), Appendix Appendix AA,, Table Table A1)A1) samples samples were were then then examined examined using using Fourier-transform Fourier-transform in- fraredinfrared spectroscopy. spectroscopy. In In the the case case of of the the diff differenterent fluorescence fluorescence intensity intensity cluster samples, three distinct secondary structures were observed.observed. The second derivative FTIR spectra of aggregates prepared at low temperature conditions (Figure3 3A,B,A,B, blueblue lines)lines) displaydisplay twotwo

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minimaminima at at 1628 1628 cm cm−−1 1andand 1615 1615 cm −11. .The The band band atat 16151615 cm cm−−11 isis associated associated with with stronger stronger hydrogenhydrogen bonding bonding within within fibril fibril beta-sheets,beta-sheets, while while the the band band at at 1628 1628 cm cm−1 −is1 is associated associated with with thethe presence presence of of weaker weaker hydrogen hydrogen bonds [[46].46]. TheThe ratioratio between between both both of of these these positions positions is is notnot identical identical for for all all samples, samples, with with the lower fluorescencefluorescence intensity intensity sample sample having having a lessa less expressedexpressed minima minima at at 1628 1628 cm cm−1,1 ,suggesting suggesting that that stronger hydrogenhydrogen bonds bonds within within the the fibril fibril resultresult in in less less bound-ThT bound-ThT or or a a lower lower fluorescencefluorescence quantum quantum yield. yield.

I 25°C A I 30°C B I 35°C C

II II II

III III III

IV IV IV

I IV Increasing fluorescence intensity 1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600

I 40°C D I 45°C E I 50°C F

II II II

III III III

IV IV IV

1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600

I 55°C G I 60°C H I 65°C I

II II II

III III III

IV IV IV

1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 Wavenumber (cm-1) Wavenumber (cm-1) Wavenumber (cm-1)

FigureFigure 3. Second 3. Second derivatives derivatives of of Fourier-transform Fourier-transform infrared spectraspectra (FTIR) (FTIR) of of prion prion protein protein fibril fibril samples samples from from their their respective respec- tiveexcitation-emission excitation-emission matrix matrix (EEM) (EEM) cluster. cluster. FTIR FTIR spectra spectra were were acquired acquired for fibrils for fibrils prepared prepared under 25under◦C(A 25), 30°C◦ (C(A),B ),30 35 °C◦ C(B), 35 °C(C), (C 40), ◦40C( °CD), ( 45D),◦ C(45 E°C), 50(E),◦C( 50F °C), 55 (F◦),C( 55G °C), 60 (G◦C(), 60H )°C and (H 65) and◦C( I65) temperature °C (I) temperature conditions. conditions. The roman The numerals roman indicatenumerals indicatethe sample the sample ThT-fluorescence ThT-fluorescence intensity, intensity, with I being with the I sample being withthe sample the lowest with emission the lowest intensity emission and IV–the intensity largest. and Spectra IV–the largest.are colour-coded Spectra are blue,colour-coded green and blue, red basedgreenon and their red main based minima on their position main similarity.minima position Dotted greysimilarity. lines indicate Dotted the grey main lines indicatespectra the positions, main spectra where position differencess, where are observed. differences are observed.

TheThe 30–65 30–65 °C◦C sample sample set set clusters clusters (Figure3 B–I)3B-I) contain contain a a second second type type of of fibrils, fibrils, with with − thethe main main second second derivative derivative FTIR FTIR spectrum minima minima located located at at 1626 1626 cm cm1,−1 which, which suggests suggests a a singlesingle dominant dominant type type of of hydrogen hydrogen bonding bonding withinwithin thethe fibrils. fibrils. This This aggregate aggregate conformation conformation waswas the the most most abundant, abundant, as as it was it was observed observed in ineight eight out out of ofthe the nine nine cluster cluster sample sample sets. sets. The The third fibril conformation was only observed at higher temperatures, namely 55 ◦C third fibril conformation◦ was only observed at higher temperatures, namely 55 °C (Figure 3G)(Figure and 603G) °Cand (Figure 60 C 3H). (Figure Similar3H). to Similar the low to thetemperature low temperature fibril conformation, fibril conformation, two minima two minima associated with beta-sheet hydrogen bonding are observed, however, unlike in the associated with beta-sheet hydrogen bonding are observed, however, unlike in the case of fibril prepared at low temperature, the second minima were at 1618 cm−1, rather than 1615 cm−1, which indicates a weaker mode of hydrogen bonding.

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case of fibril prepared at low temperature, the second minima were at 1618 cm−1, rather than 1615 cm−1, which indicates a weaker mode of hydrogen bonding. When the outlier fibril samples (Figure2 (red), AppendixA, Table A1) were replicated and centrifuged, certain sample aggregate pellets were semitransparent and gel-like. Coincidentally, their FTIR spectra displayed a noticeable band associated with guanidine hydrochloride, despite multiple centrifugation and resuspension steps. Such abnormalities were only observed in the case of fibrils prepared under 25 ◦C and 30 ◦C temperatures. One likely explanation for this event is that this type of fibril conformation is capable of trapping some of the denaturant present in solution during aggregation, either in fibril cavities or by associating into this gel-like structure. While this, in itself, indicates a different type of aggregate, in order to acquire comparable FTIR spectra, the samples had to be washed three additional times (the second derivative FTIR spectra of these gel-like samples are marked with a * symbol in Figure4). Certain low-temperature samples also contain a very Int. J. Mol. Sci. 2021, 22, x − 7 of 18 small band at 1604 cm 1, which means that they may contain a minor concentration of GuHCl as well.

I 25°C A 30°C B I 35°C C I* II* II II* III* III III IV IV IV

1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600

40°C D 45°C E I 50°C F I I II II II III III III IV IV IV

1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 55°C G 60°C H 65°C I I I I

II II II III III III IV IV IV

1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 1700 1680 1660 1640 1620 1600 Wavenumber (cm-1) Wavenumber (cm-1) Wavenumber (cm-1)

FigureFigure 4. Second 4. Second derivatives derivatives of Fourier-transform of Fourier-transform infrared infrared spectra spectra (FTIR) (FTIR) of prion of prion protein protein fibril ﬁbril samples, samples, which which were were outliersoutliers Figure. Figure. 25 °C 25 (A◦C(), 30A ),°C 30 (B◦C(), 35B), °C 35 (◦CC(), C40), 40°C ◦(C(D),D 45), 45°C◦ C((E),E ),50 50 °C◦C( (FF),), 55 55 °C◦C( (GG),), 60 60 ◦°CC( (H)) andand 65 65◦ C(°C I()I) temperature temperature conditions.conditions. The The Roman Roman numerals numerals indicate indicate the the outlier outlier sample sample (list (list of ofoutlier outlier sample sample excitation-emission excitation-emission matrix matrix (EEM) (EEM) po- sitionspositions is located is located at the at Appendix the Appendix A TableA Table A1). A1 ).Spectra Spectra that that sh shareare similarities similarities to the mainmain clustercluster samples samples are are colour-coded colour-coded accordinglyaccordingly (blue, (blue, green green and and red), red), while while black black spectra spectra are are unique unique outliers. outliers. The The second second derivative derivative FTIR FTIR spectra spectra of ofthese these gel- likegel-like samples samples are marked are marked with witha *. a *.

Interestingly, there were four outliers that did not resemble any of the three main fibril conformations. The outlier generated at 25 °C (Figure 4A, black line) had a more significant minimum at 1664 cm−1, which is associated with the presence of turn/loop motifs in the fibril structure [46]. It also had two minima at 1622 cm−1 and 1615 cm−1, which means that, along with the strong type of hydrogen bonding, there was also a type of bonding that was dissimilar to all other fibrils. The outlier generated at 40 °C (Figure 4D, black line) shares some similarities to the high-temperature conformation, however, it has more minima associated with turn/loop motifs. The outlier generated at 55 °C (Figure 4G, black line) had a minimum at 1664 cm−1, similarly to the 25 °C outlier, however, it has the most intense band at 1618 cm−1 with a smaller minimum at 1630 cm−1, which means that the fibril structure had both a large amount of strong hydrogen bonds between beta- sheets, and some weak bonding. The 60 °C outlier (Figure 4H) had similarities to the 55 °C outlier, with both having significant minima at 1664 cm−1, however, the 1630 cm−1 position is more of a shoulder, rather than a minimum, which indicates slightly less weak hydrogen bonds (these two samples were regarded as similar outliers). All four samples that had a gel-like appearance (marked with * in Figure 4) share minima positions with

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The outlier sample second derivative FTIR spectra (Figure4) were significantly more diverse than in the case of the main cluster samples. While most spectra shared similar minima positions to the three cluster fibril conformation spectra (Figure4A–I), color-coded accordingly), the band intensity ratios experienced a considerably larger variation, which suggests the presence of multiple aggregates with distinct secondary structures. This is best seen in the case of 45 ◦C outliers (Figure4E), where all spectra minima had similar positions, yet the 1628 cm−1 and 1618 cm−1 minima ratios were different. There was also a lot less temperature-dependent conformation distribution, as the structure associated with fibrils prepared under high temperatures was observed in the 35 ◦C outlier sample (Figure4C), as was the structure associated with fibrils prepared under low temperatures. Interestingly, there were four outliers that did not resemble any of the three main fibril conformations. The outlier generated at 25 ◦C (Figure4A, black line) had a more significant minimum at 1664 cm−1, which is associated with the presence of turn/loop motifs in the fibril structure [46]. It also had two minima at 1622 cm−1 and 1615 cm−1, which means that, along with the strong type of hydrogen bonding, there was also a type of bonding that was dissimilar to all other fibrils. The outlier generated at 40 ◦C (Figure4D, black line) shares some similarities to the high-temperature conformation, however, it has more minima associated with turn/loop motifs. The outlier generated at 55 ◦C(Figure4G , black line) had a minimum at 1664 cm−1, similarly to the 25 ◦C outlier, however, it has the most intense band at 1618 cm−1 with a smaller minimum at 1630 cm−1, which means that the fibril structure had both a large amount of strong hydrogen bonds between beta-sheets, and some weak bonding. The 60 ◦C outlier (Figure4H) had similarities to the 55 ◦C outlier, with both having significant minima at 1664 cm−1, however, the 1630 cm−1 position is more of a shoulder, rather than a minimum, which indicates slightly less weak hydrogen bonds (these two samples were regarded as similar outliers). All four samples that had a gel-like appearance (marked with * in Figure4) share minima positions with the high temperature conformation, however, their FTIR second derivative spectra minima are far more expressed when compared to their high-temperature counterparts, while the turn/loop motif minima are less expressed, suggesting a slightly different secondary structure. The seven samples, which showed significant variations in their second derivative FTIR spectra, were further examined by atomic force microscopy. The first notable factor was the difference in fibril self-association (Figure5A–G, AppendixA, Figures A2A–G and A3A–G). The 25 ◦C Cluster II, 25 ◦C Outlier I, 25 ◦C Outlier II, 45 ◦C Cluster II and 60 ◦C Cluster I samples contained aggregates that were highly prone towards binding to one another and forming large fibril clumps, while all other samples were more disperse. Despite the vigorous agitation during aggregation, the 25 ◦C Outlier II, 40 ◦C Outlier IV and 60 ◦C Cluster I samples had relatively long fibrils, suggesting a higher structural stability. Due to certain samples forming large superstructural clusters, conducting a statistical analysis was only possible on their height. A one-way ANOVA Bonferroni means comparison of the height data (n = 50, significance level–0.01) revealed that only the 60 ◦C Cluster I sample had a significantly different height distribution from all other samples, except for the 25 ◦C Outlier II sample (Figure5H). Int. J. Mol. Sci. Sci. 20212021,, 22,, x 5075 9 8of of 18 16

Figure 5. Atomic force microscopy images of prion protein fibrilsfibrils possessing distinct Fourier-transform infrared (FTIR) spectra ((A–G), conditions and sample type shown above images). Fibril height (H) distribution, where box plots indicate spectra ((A–G), conditions and sample type shown above images). Fibril height (H) distribution, where box plots indicate the interquartile range and error bars are one standard deviation (n = 50). Fibril height values were obtained by tracing the interquartile range and error bars are one standard deviation (n = 50). Fibril height values were obtained by tracing perpendicular to each fibril’s axis (only separate, non-clumped aggregates were examined). perpendicular to each fibril’s axis (only separate, non-clumped aggregates were examined). 3. Discussion 3. Discussion Considering that a shift in sample secondary structure and the existence of high in- Considering that a shift in sample secondary structure and the existence of high inten- tensity samples coincide with the temperature, at which the prion protein switches be- sity samples coincide with the temperature, at which the prion protein switches between tween the folded and unfolded states, suggests that this factor is important in determining the folded and unfolded states, suggests that this factor is important in determining the the type of fibril conformation. Since the temperatures under which the protein is in its type of fibril conformation. Since the temperatures under which the protein is in its mostly mostlyfolded statefolded also state result also in result significantly in significantly slower aggregation,slower aggregation, this provides this provides two possibilities. two possibilities.First, certain First, nuclei certain may nuclei require may a relatively require along relatively time to long form time and to such form stable and aggregationsuch stable aggregationcenters simply centers do not simply have do the not required have the time required to assemble time to duringassemble quick during fibrillization quick fibril- at lizationhigher temperatures.at higher temperatures. Alternatively, Alternatively, it is also possible it is also that possible distinct that aggregates distinct aggregates require a requirespecific a semifolded specific semifolded state of the state protein of the to protein both form to both and form grow, and which grow, makes which their makes nu-

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cleation/elongation events impossible when most of the protein is in a fully unfolded state. In addition, the tested prion protein (89–230 sequence with His-tag) had 54 polar amino acids (33% of the full sequence), 32 of which were charged, which may contribute to the appearance of distinct hydrogen bonds from polar–polar interactions under certain temperature conditions [47]. While such a temperature-dependent shift in fibril conformations can be expected, the truly peculiar aspect is the variation in aggregate secondary structure and morphology. Not a single sample set in the entire tested temperature range contained a well-defined and homogenous collection of fibrils. Each condition resulted in a variable bound-ThT fluorescence intensity, which was well beyond a standard deviation, different EEM maxima positions, FTIR spectra and even fibril morphologies. The large variation of all these factors suggests that prion proteins can form different conformation fibrils or non-homogenous aggregate mixtures under all tested temperatures and this event may encompass other conditions as well. In essence, this means that the assumption of identical conditions leading to identical samples is not correct for prion proteins and this factor has to be taken into account during assays which involve these proteins. Another interesting observation is the existence of outliers in every set of samples. These outliers are few in number, suggesting that their aggregation pathways are either very complex or unlikely under the given conditions, however, they possess significantly different characteristics. Some of them are so distinct from other fibrils that they can be identified even with a simple visual inspection. While all other samples resulted in an opaque pellet after centrifugation, one type of outlier fibrils had a transparent, gel-like appearance and had a higher tendency of entrapping the buffer solution within itself. Other outliers seem to possess higher structural stabilities, as seen from AFM images. The differences in ThT binding characteristics also suggest a different surface morphology or charge, especially in the case of high fluorescence intensity samples. Taking everything into consideration, it appears that the environmental conditions during prion protein aggregation do not have a strict control over the type of fibril that forms, but rather determine the dominant aggregate conformation with a certain level of variability. None of the tested conditions resulted in homogenous samples and this factor has to be taken into account, especially when conducting screenings for antiamyloid compounds, as different fibril types may have distinct responses to certain drug molecules.

4. Materials and Methods 4.1. Prion Protein Aggregation Mouse recombinant prion protein (89–230) was purified as described previously [43], without the His-tag cleavage step, dialyzed in 10 mM sodium acetate (pH 4.0) for 24 h, concentrated to 3.0 mg/mL and stored at −80 ◦C prior to use. The protein solution was mixed with 50 mM sodium phosphate (pH 6.0) buffers, containing 0 M or 6 M guanidine hydrochloride (GuHCl) and a ThT stock solution (10 mM) to a final reaction solution, containing 2 M GuHCl, 100 µM ThT and 0.5 mg/mL protein concentration. The reaction solutions were distributed to 96-well half-area non-binding plates (cat. No 3881, Fisher Scientific, USA, Hampton, NH, USA) (100 µL final volume, each well contained a 3 mm bead), which were sealed with a Nunc sealing tape. The samples were incubated at a set temperature (range from 25 to 65 ◦C), with constant 600 RPM agitation (vigorous, fragmentation-inducing agitation was used to achieve complete fibrillization in a reasonable timeframe). Sample fluorescence was scanned every 5 min, using an excitation wavelength of 440 nm and an emission wavelength of 480 nm. Due to the stochastic nature of aggregation, a small number of samples displayed unusual aggregation kinetics (no detectable signal or signal jumps), which made it impossible to determine their kinetic parameters. Due to this reason, 6 samples were removed from every 96-well plate. The aggregation lag time (tlag) was calculated by applying a sigmoidal Boltzmann equation fit to the data. An example is provided as AppendixA, Figure A4. All aggregation kinetic data is available as Supplementary Material. Int. J. Mol. Sci. 2021, 22, 5075 10 of 16

4.2. ThT-Assay After aggregation had occurred, each sample was taken out of the 96-well plate and each well was additionally washed with the reaction solution (50 mM sodium phosphate buffer (pH 6.0) with 2 M GuHCl and 100 µM ThT) in order to collect all the fibrils. The samples were then further diluted using the reaction buffer solution to a final volume of 500 µL (5-fold dilution). Samples were then sonicated for 30 s using a Bandelin Sonopuls (Berlin, Germany) Ultrasonic homogenizer, equipped with a MS-72 tip (20% power). Each sample‘s excitation–emission matrix (EEM) was then scanned using a Varian Cary Eclipse (Agilent Technologies, Santa Clara, CA, USA) fluorescence spectrophotometer (excitation wavelength range was from 435 to 465 nm, emission wavelength range—from 460 to 500 nm; both excitation and emission slit widths were 5 nm) at 25 ◦C. Absorbance spectra were acquired from 300 to 600 nm using a Shimadzu (Kyoto, Japan) UV-1800 spectrophotometer. EEMs were then corrected for the inner filter effect and their signal intensity centre of mass was calculated as described previously [48]. All EEM data is available as Supplementary Material. Sample maximum fluorescence intensity distributions were compared using a one-way ANOVA Bonferroni means comparison with a significance level of 0.01 (Origin 2018 software, OriginLab Corporation, Northampton, MA, USA).

4.3. Fourier-Transform Infrared Spectroscopy In order to obtain a larger quantity of fibrils for higher quality FTIR spectra, each sample was combined with a non-aggregated protein solution (identical to the one used for initial aggregation) in equal volumes. Final reaction solutions contained 0.25 mg/mL prion protein and 0.05 mg/mL fibrils (assuming a 100% fibrillization and complete aggregate recovery from wells). The lower non-aggregated protein concentration was used to minimize nucleation events and the relatively high concentration of fibrils assured quick self-replication [24]. The reaction solutions were then incubated under conditions, which were identical to their respective aggregate preparation conditions, in order to achieve effective self-replication. The resulting aggregate solutions were centrifuged for 30 min at 10,000× g, after which the supernatant was removed and the fibril pellet was resuspended into 250 µLD2O (D2O was supplemented with 100 mM NaCl, which improves fibril sedimentation [49]). This centrifugation and resuspension procedure was repeated 4 times. After the final centrifugation step, the pellet was resuspended into 50 µLD2O. The FTIR spectra were acquired as described previously [28]. A D2O spectrum was subtracted from each sample spectrum, after which they were baseline corrected and normalized between 1595 and 1700 cm−1. All data processing was done using GRAMS software and all FTIR data is available as Supplementary Material.

4.4. Atomic Force Microscopy Fibril samples were agitated by mixing for 30 s in order to reduce aggregate clumping. Afterwards, 20 µL of each sample was deposited on freshly-cleaved mica and left to adsorb for 60 s. Then the mica were rinsed with 2 mL of H2O and air-dried. AFM images were then acquired as previously described [28]. In short, 1024 pixel × 1024 pixel resolution three- dimensional maps were obtained for each sample using a dimension icon (Bruker, Billerica, MA, USA) atomic force microscope. The images were then flattened using Gwyddion 2.5.5 software. Fibril height was determined from line profiles taken perpendicular to the fibril axes. Fibril height distribution was compared using a one-way ANOVA Bonferroni means comparison with a significance level of 0.01 (Origin 2018 software, OriginLab Corporation, Northampton, MA, USA).

Supplementary Materials: Supplementary Materials can be found at https://www.mdpi.com/ article/10.3390/ijms22105075/s1. Supplementary Materials contain the following: raw data of prion protein aggregation, FTIR spectra of prion protein ﬁbrils, excitation-emission matrices of ﬁbril-bound ThT and atomic force microscopy images in TIF format. Int. J. Mol. Sci. 2021, 22, 5075 11 of 16

Author Contributions: Conceptualization, M.Z., A.S. and V.S.; investigation, M.Z., A.S., K.M. and R.S.; resources, V.S.; writing—original draft preparation, M.Z.; writing—review and editing, M.Z., A.S. and V.S.; supervision, V.S.; funding acquisition, V.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the grant no. S-SEN-20-3 from the Research Council of Lithuania. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in Supplementary Material. Acknowledgments: The authors acknowledge G. Niaura from the Center of Physical Sciences and Technology for the access to FTIR. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Appendix A

Table A1. Excitation-emission matrix maximum position wavelengths (nm) and emission intensities of cluster and outlier samples.

T Cluster Excitation Emission Intensity Outliers Excitation Emission Intensity 25 ◦C I 444.1 483.7 98.0 I 445.0 482.3 61.1 25 ◦C II 444.3 484.1 145.6 II 446.4 486.1 440.6 25 ◦C III 443.0 483.9 154.7 III 447.4 485.3 594.5 25 ◦C IV 445.9 484.3 200.6 IV 446.8 483.6 187.6 30 ◦C I 443.5 484.3 113.7 I 446.8 486.3 587.1 30 ◦C II 444.2 483.9 164.8 II 447.2 485.4 590.8 30 ◦C III 446.2 483.1 198.6 III 441.8 484.1 144.4 30 ◦C IV 448.7 483.1 251.7 IV 444.9 482.8 201.4 35 ◦C I 445.7 483.7 180.5 I 451.5 482.6 183.9 35 ◦C II 446.2 483.4 210.9 II 446.8 485.9 465.7 35 ◦C III 446.3 483.8 220.9 III 443.7 483.8 154.6 35 ◦C IV 446.8 483.8 271.4 IV 450.1 483.4 236.4 40 ◦C I 446.1 483.3 71.4 I 446.2 485.4 293.8 40 ◦C II 448.0 483.3 207.0 II 442.8 483.5 38.8 40 ◦C III 447.8 482.9 242.1 III 452.0 482.4 175.3 40 ◦C IV 446.3 483.4 274.4 IV 457.8 482.9 141.3 45 ◦C I 445.9 483.0 117.1 I 454.1 482.3 132.6 45 ◦C II 447.1 483.0 212.0 II 450.8 482.9 142.8 45 ◦C III 445.1 483.2 224.2 III 442.4 485.1 142.4 45 ◦C IV 447.5 483.7 250.1 IV 442.7 484.3 111.8 50 ◦C I 443.2 483.4 49.9 I 455.6 482.6 88.9 50 ◦C II 444.6 483.3 107.6 II 447.1 485.1 404.2 50 ◦C III 446.0 483.2 175.8 III 439.7 484.6 26.1 50 ◦C IV 447.1 482.9 273.2 IV 442.5 486.3 79.3 55 ◦C I 442.8 483.8 70.3 I 441.8 486.8 104.7 Int. J. Mol. Sci. 2021, 22, 5075 12 of 16

Int. J. Mol. Sci. 2021, 22, x Table A1. Cont. 13 of 18

T Cluster Excitation Emission Intensity Outliers Excitation Emission Intensity 55 ◦C II 443.7 482.9 112.2 II 443.0 485.5 119.2 55 °C III 445.655 ◦C III482.7 445.6170.5 482.7III 170.5449.7 III 449.7482.5 482.5158.8 158.8 ◦ 55 °C IV 446.455 C IV483.6 446.4277.7 483.6IV 277.7441.6 IV 441.6483.7 483.762.8 62.8 ◦ 60 °C I 442.860 C I482.9 442.857.9 482.9I 57.9442.2 I 442.2485.8 485.8122.7 122.7 ◦ 60 °C II 444.760 C II483.2 444.792.2 483.2II 92.2448.9 II 448.9484.6 484.6108.3 108.3 ◦ 60 °C III 443.960 C III483.8 443.9117.2 483.8III 117.2450.1 III 450.1482.4 482.4166.6 166.6 60 ◦C IV 448.9 482.8 295.2 IV 442.1 483.9 106.6 60 °C IV 448.9 482.8 295.2 IV 442.1 483.9 106.6 65 ◦C I 444.9 483.4 30.0 I 439.4 483.9 29.7 65 °C I 444.9 483.4 30.0 I 439.4 483.9 29.7 65 ◦C II 446.6 482.8 146.3 II 443.9 484.5 112.0 65 °C II 446.6 482.8 146.3 II 443.9 484.5 112.0 65 ◦C III 447.3 482.7 201.9 III 447.8 481.9 219.1 65 °C III 447.365 ◦C IV482.7 448.6201.9 482.7III 305.8447.8 IV 447.9481.9 483.9219.1 127.6 65 °C IV 448.6 482.7 305.8 IV 447.9 483.9 127.6

35 45 Outliers: 14 I A B C 40 II 30 III 12 35 IV 25 Cluster samples: 10 30 I II 20 25 III 8 IV 15 20 6 15 4 10 10 2 5 5

Fluorescence intensity (a.u.) intensity Fluorescence 0 0 0

0 100 200 300 400 500 600 0 100 200 300 400 500 0 50 100 150 200 250 300 350

4 DE16 F 15 14 3 12 10 10 2 8

5 6

1 4

0 2 Fluorescence intensity (a.u.) intensity Fluorescence 0 0 0 50 100 150 200 250 0 25 50 75 100 125 150 0 25 50 75 100 125 150

3 G 5 H I 4

4 2 3 3

2 2 1

1 1 0

Fluorescence intensity (a.u.) intensity Fluorescence 0 0 0 25 50 75 100 125 0 25 50 75 100 125 0255075100 Time (min) Time (min) Time (min) ◦ ◦ ◦ FigureFigure A1. ReseedingA1. Reseeding aggregation aggregation kinetics of kinetics the selected of clusterthe selected and outlier cluster samples and under outlier 25 samplesC(A), 30 C(underB), 35 25C( °CC ), ◦ ◦ ◦ ◦ ◦ ◦ 40 (AC(),D 30), 45 °CC( (BE),), 35 50 °CC( (FC),), 55 40C( °CG ),(D 60), 45C( H°C) and(E), 65 50C( °CI )( temperatureF), 55 °C (G conditions.), 60 °C (H) and 65 °C (I) temperature conditions.

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FigureFigure A2. First A2. First set of set larger of larger scale scale atomic atomic force force microscopy microscopy imagesimages ofof prion prion protein protein ﬁbrils fibrils possessing possessing distinct distinct Fourier- Fourier- transformtransform infrared infrared (FTIR) (FTIR) spectra spectra ((A ((–AG–G), ),conditions conditions and samplesample type type shown shown above above images). images).

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FigureFigure A3. Second A3. Second set of set larger of larger scale scale atomic atomic force force microscopy microscopy images ofof prion prion protein protein ﬁbrils fibrils possessing possessing distinct distinct Fourier- Fourier- transformtransform infrared infrared (FTIR) (FTIR) spectra spectra ((A ((–AG–G), ),conditions conditions and and sample type type shown shown above above images). images).

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tlag 600

500

y = A2 A1 – initial value 400 A2 – final value x0 –center 300 dx – time constant t –lag time lag 200 x0 = (A1+A2)/2 t50 – time when y = x0 100 Fluorescence intensity (a.u.) intensity Fluorescence

y = A1 y’ = (A2-A1)/4dx 0

0 250 500 750 1000 1250 1500 1750 Time (min)

Figure A4. Boltzmann sigmoidalsigmoidal equationequation fit fit example example and and tlag tlagcalculation. calculation. Data Data points points were were obtained ob- bytained tracking by tracking the fluorescence the fluorescence intensity intensity of thioflavin-T of thioflavin-T during during prionprotein prion protein aggregation. aggregation. Horizontal blueHorizontal lines indicate blue lines the indicate lowest, intermediate the lowest, intermediate and highest averageand highest fluorescence average intensity.fluorescence The intensity. red line is aThe Boltzmann red line is sigmoidal a Boltzmann equation sigmoidal fit (with equation a blue fit tangent (with linea blue at thetangent intermediate line at the value intermediate of the fit). value of the fit). References 1.ReferencesChiti, F.; Dobson, C.M. Protein Misfolding, Amyloid Formation, and Human Disease: A Summary of Progress Over the Last 1. Decade.Chiti, F.;Annu. Dobson, Rev. C.M. Biochem. 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